Ucp5

ABSTRACT

The present invention is directed to novel polypeptides having homology to certain human uncoupling proteins (“UCPs”) and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention, and methods for producing the polypeptides of the present invention.

RELATED APPLICATIONS

This application is a non-provisional application filed under 37 CFR1.53(b) claiming priority under Section 119 to provisional applicationNos. 60/110,286 filed Nov. 30, 1998, 60/129,583 filed Apr. 16, 1999, and60/143,886 filed Jul. 15, 1999, the contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA having homology to certain human uncouplingproteins, and to the recombinant production of novel polypeptides,designated herein as “uncoupling protein 5” or “UCP5.”

BACKGROUND OF THE INVENTION

Uncoupling proteins or “UCPs”, believed to play a role in the metabolicprocess, have been reported in the literature. UCPs were first found anddescribed in the brown fat cells of hibernating animals, such as bears.UCPs were believed to help such hibernators and other cold-weatheradapted animals maintain core body temperatures in cold weather byraising their body's resting metabolic rate. Because humans possessrelatively small quantities of brown adipose tissue, UCPs wereoriginally thought to play a minor role in human metabolism.

Several different human uncoupling proteins have now been described.[See, generally, Gura, Science, 280:1369-1370 (1998)]. The humanuncoupling protein referred to as UCP1 was identified by Nicholls et al.Nicholls et al. showed that the inner membrane of brown fat cellmitochondria was very permeable to proteins, and the investigatorstraced the observed permeability to a protein, called UCP1, in themitochondrial membrane. Nicholls et al. reported that the UCP1, bycreating such permeability, reduced the number of ATPs that can be madefrom a food source, thus raising body metabolic rate and generatingheat. [Nicholls et al., Physiol. Rev., 64, 1-64 (1984)].

It was later found that UCP1 is indeed expressed only in brown adiposetissue [Bouillaud et al., Proc. Natl. Acad. Sci., 82:445-448 (1985);Jacobsson et al., J. Biol. Chem., 260:16250-16254 (1985)]. Geneticmapping studies have shown that the human UCP1 gene is located onchromosome 4. [Cassard et al., J. Cell. Biochem., 43:255-264 (1990)].UCP1 recently has been called thermogenin. [Palou et al., Int. J.Biochem. & Cell Bio., 30: 7-11 (1998)]. Palou et al. describe that UCP1synthesis and activity are regulated by norepinephrine. [Palou et al.,supra].

Another human UCP, referred to as C5 or UCPH or UCP2, has also beendescribed. [Gimeno et al., Diabetes, 46:900-906 (1997); Fleury et al.,Nat. Genet., 15:269-272 (1997); Boss et al., FEBS Letters, 408:39-42(1997); see also, Wolf, Nutr. Rev., 55:178-179 (1997); U.S. Pat. No.5,702,902]. Fleury et al. teach that the UCP2 protein has 59% amino acididentity to UCP1, and that UCP2 maps to regions of human chromosome 11which have been linked to hyperinsulinaemia and obesity. [Fleury et al.,supra]. It has also been reported that UCP2 is expressed in a variety ofadult tissues, such as brain and muscle and fat cells. [Gimeno et al.,supra, and Fleury et al., supra]. Similarly, U.S. Pat. No. 5,702,902reported a relatively complex pattern of tissue distribution, with mRNAaccumulation appearing to be greatest in muscle tissue.

A third human UCP, UCP3, was recently described in Boss et al., supra;Vidal-Puig et al., Biochem. Biophys. Res. Comm., 235:79-82 (1997);Solanes et al., J. Biol. Chem., 272:25433-25436 (1997); and Gong et al.,J. Biol. Chem., 272:24129-24132 (1997). [See also Great Britain PatentNo. 9716886]. Solanes et al. report that unlike UCP1 and UCP2, UCP3 isexpressed preferentially in human skeletal muscle, and that the UCP3gene maps to human chromosome 11, adjacent to the UCP2 gene. [Solanes etal., supra]. Gong et al. describe that the UCP3 expression can beregulated by known thermogenic stimuli, such as thyroid hormone,beta3-andrenergic agonists and leptin. [Gong et al., supra].

UCP1, UCP2, and UCP3 share several characteristics with mitochondrialmembrane transporters. [Boss et al., Euro. J. Endocrinology, 139: 1-9(1998)]. All three UCPs are about 300 amino acids long and have amolecular mass of about 30 kDa. [Boss et al., supra]. Each also hasthree typical mitochondrial energy transfer protein signatures. [Boss etal., supra].

SUMMARY OF THE INVENTION

A cDNA clone (DNA 80562-1663) has been identified, having certainhomologies to some known human uncoupling proteins, that encodes a novelpolypeptide, designated in the present application as “UCP5.”

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a UCP5 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a UCP5 polypeptide comprising the sequence of amino acidresidues from about 1 to about 325, inclusive of FIG. 1 (SEQ ID NO: 1),or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a UCP5 polypeptide comprising DNA hybridizing to thecomplement of the nucleic acid between about nucleotides 10 and about987 inclusive, of FIG. 1 (SEQ ID NO: 2). Preferably, hybridizationoccurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No.203325, or (b) the complement of the DNA molecule of (a). In a preferredembodiment, the nucleic acid comprises a DNA encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No.203325.

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues from about 1 to about 325, inclusive of FIG. 1 (SEQ ID NO:1), or the complement of the DNA of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1to about 325, inclusive of FIG. 1 (SEQ ID NO: 1), or (b) the complementof the DNA of (a).

Further embodiments of the invention are directed to fragments of theUCP5 coding sequence, which are sufficiently long to be used ashybridization probes. Preferably, such fragments contain at least about20 to about 80 consecutive bases included in the sequence of FIG. 1 (SEQID NO: 2). Optionally, such fragments include the N-terminus or theC-terminus of the sequence of FIG. 1 (SEQ ID NO: 2).

In another embodiment, the invention provides a vector comprising DNAencoding UCP5 or its variants. The vector may comprise any of theisolated nucleic acid molecules hereinabove defined.

A host cell comprising such a vector is also provided. By way ofexample, the host cells may be CHO cells, E. coli, or yeast. A processfor producing UCP5 polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of UCP5and recovering UCP5 from the cell culture.

In another embodiment, the invention provides isolated UCP5 polypeptideencoded by any of the isolated nucleic acid sequences hereinabovedefined.

In a specific aspect, the invention provides isolated native sequenceUCP5 polypeptide, which in one embodiment, includes an amino acidsequence comprising residues 1 to 325 or residues 20 to 325 of FIG. 1(SEQ ID NO: 1).

In another aspect, the invention concerns an isolated UCP5 polypeptide,comprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 to 325 or residues 20 to 325, of FIG. 1 (SEQ ID NO: 1).

In a further aspect, the invention concerns an isolated UCP5polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 to325 or residues 20 to 325 of FIG. 1 (SEQ ID NO: 1).

In yet another aspect, the invention concerns an isolated UCP5polypeptide, comprising the sequence of amino acid residues 1 to about325 or residues 20 to 325, of FIG. 1 (SEQ ID NO: 1), or a fragmentthereof sufficient to, for instance, provide a binding site for ananti-UCP5 antibody. Preferably, the UCP5 fragment retains at least onebiological activity of a native UCP5 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a UCP5 polypeptide comprising the sequenceof amino acid residues from about 1 to about 325 of FIG. 1 (SEQ ID NO:1), or (b) the complement of the DNA molecule of (a), and if the testDNA molecule has at least about an 80% sequence identity, preferably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), (ii) culturing a host cell comprising the testDNA molecule under conditions suitable for expression of thepolypeptide, and (iii) recovering the polypeptide from the cell culture.

In another embodiment, the invention provides chimeric moleculescomprising a UCP5 polypeptide fused to a heterologous polypeptide oramino acid sequence. An example of such a chimeric molecule comprises aUCP5 polypeptide fused to an epitope tag sequence or a Fc region of animmunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to UCP5 polypeptide. Optionally, the antibody is amonoclonal antibody.

In yet another embodiment, the invention concerns agonists andantagonists of the native UCP5 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-UCP5 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native UCP5 polypeptide, by contacting thenative UCP5 polypeptide with a candidate molecule and monitoring thedesired activity. The invention also provides therapeutic methods anddiagnostic methods using UCP5.

In a still further embodiment, the invention concerns a compositioncomprising a UCP5 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of a cDNA encoding native sequenceUCP5 (SEQ ID NO: 2), the complement of the nucleotide sequence of a cDNAencoding native sequence UCP5 (SEQ ID NO: 32), and the correspondingderived amino acid sequence of a native sequence UCP5 (SEQ ID NO: 1).

FIG. 2 shows an amino acid sequence alignment of UCP5 with otheruncoupling proteins, UCP1 (SEQ ID NO: 33), UCP2 (SEQ ID NO: 34), UCP3(SEQ ID NO: 35), UCP4 (SEQ ID NO: 36), and UCP5 (SEQ ID NO: 1). The sixputative transmembrane domains are shown and are underlined (and labeledI to VI, respectively). The asterisks (*) shown below the proteinsequence indicate three (3) putative mitochondrial carrier proteinmotifs. A putative nucleotide binding domain is double underlined.

FIGS. 3A-3G show the results of Northern blot analysis. Human adulttissues (A-C), cancer cell lines (D), human adult brain tissue (E, F)and mouse multiple tissues (G) (Clontech) were probed with human ormouse UCP5 cDNA.

FIGS. 3H-3I show the results of real time quantitative RT-PCR assaysperformed using primers and probes with specificities toward total UCP5,mUCP5L, or UCP5SI, and using RNA from various human (H) and murine (I)tissues.

FIGS. 4A-4F show the results of in vitro assays conducted to determinethe effects of UCP5 expression on mitochondrial membrane potential.

FIG. 5 shows a “from DNA” sequence (SEQ ID NO: 5) assembled fromselected EST sequences.

FIGS. 6A-6C show the results of in vitro assays conducted to determinethe effect of food consumption on the expression of UCP5 mRNA in braintissue.

FIGS. 7A-7C show the results of in vitro assays conducted to determinethe effect of food consumption on the expression of UCP5 mRNA in livertissue.

FIGS. 8A-8D show the results of in vitro assays conducted to determinethe effect of fat consumption on the expression of UCP5 mRNA in braintissue.

FIGS. 9A-9D show the results of in vitro assays conducted to determinethe effect of fat consumption on the expression of UCP5 mRNA in livertissue.

FIGS. 10A-10G show the results of in vitro assays conducted to determinethe effect of temperature stress on the expression of UCP5 mRNA in braintissue.

FIGS. 11A-11G show the results of in vitro assays conducted to determinethe effect of temperature stress on the expression of UCP5 mRNA in livertissue.

FIG. 12 shows the nucleotide sequence of a cDNA encoding hUCP5S (SEQ IDNO: 6).

FIG. 13 shows the nucleotide sequence of a cDNA encoding hUCP5SI (SEQ IDNO: 8).

FIG. 14 shows the nucleotide sequence of a cDNA encoding mUCP5S (SEQ IDNO: 10).

FIG. 15 shows the nucleotide sequence of a cDNA encoding mUCP5L (SEQ IDNO: 12).

FIG. 16 shows an amino acid sequence alignment of isoforms of UCP5,hUCP5L (SEQ ID NO: 1), hUCP5S (SEQ ID NO: 7), hUCP5SI (SEQ ID NO: 9),mUCP5L (SEQ ID NO: 13), and mUCP5S (SEQ ID NO: 11). The six putativetransmembrane domains are shown and are underlined (and labeled I to VI,respectively). The asterisks (*) shown below the protein sequenceindicates putative mitochondrial carrier protein motifs. A putativenucleotide binding domain is double underlined.

FIGS. 17A-17C show results of in vitro assays conducted to determine theeffect of UCP5 expression on mitochondrial membrane potential.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms “UCP5 polypeptide”, “UCP5 protein” and “UCP5” when used hereinencompass native sequence UCP5 and UCP5 variants (which are furtherdefined herein). The UCP5 may be isolated from a variety of sources,such as from human tissue types or from another source, or prepared byrecombinant and/or synthetic methods.

A “native sequence UCP5” comprises a polypeptide having the same aminoacid sequence as a UCP5 derived from nature. Such native sequence UCP5can be isolated from nature or can be produced by recombinant and/orsynthetic means. The term “native sequence UCP5” specificallyencompasses naturally-occurring truncated forms or isoforms,naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the UCP5. In one embodimentof the invention, the native sequence UCP5 is a mature or full-lengthhuman native sequence UCP5 (“hUCP5L”) comprising amino acids 1 to 325 ofFIG. 1 (SEQ ID NO: 1).

“UCP5 variant” means anything other than a native sequence UCP5, andincludes UCP5 having at least about 80% amino acid sequence identitywith the amino acid sequence of residues 1 to 325 of the UCP5polypeptide having the deduced amino acid sequence shown in FIG. 1 (SEQID NO: 1). Such UCP5 variants include, for instance, UCP5 polypeptideswherein one or more amino acid residues are added, or deleted, at the N-or C-terminus, as well as within one or more internal domains, of thesequence of FIG. 1 (SEQ ID NO: 1). Ordinarily, a UCP5 variant will haveat least about 80% amino acid sequence identity, more preferably atleast about 85% amino acid sequence identity, even more preferably atleast about 90% amino acid sequence identity, and most preferably atleast about 95% sequence identity with the amino acid sequence ofresidues 1 to 325 of FIG. 1 (SEQ ID NO: 1).

The term “hUCP5S” as used herein refers to the polypeptide identifiedfrom human tissue comprising the amino acid sequence of FIG. 16 (SEQ IDNO: 7).

The term “hUCP5SI” as used herein refers to the polypeptide identifiedfrom human tissue comprising the amino acid sequence of FIG. 16 (SEQ IDNO: 9).

The term “mUCP5L” as used herein refers to the polypeptide identifiedfrom murine tissue comprising the amino acid sequence of FIG. 16 (SEQ IDNO: 13).

The term “mUCP5S” as used herein refers to the polypeptide identifiedfrom murine tissue comprising the amino acid sequence of FIG. 16 (SEQ IDNO: 11).

“Percent (%) amino acid sequence identity” with respect to the UCP5sequences identified herein is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the UCP5 sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. % identity can be determined by WU-BLAST-2,obtained from [Altschul et al., Methods in Enzymology, 266: 460-480(1996); http://blast.wustl/edu/blast/README.html]. WU-BLAST-2 usesseveral search parameters, most of which are set to the default values.The adjustable parameters are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSPS2 parameters are dynamic values and are established by the programitself depending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. A % amino acid sequence identity value isdetermined by the number of matching identical residues divided by thetotal number of residues of the “longer” sequence in the aligned region.The “longer” sequence is the one having the most actual residues in thealigned region (gaps introduced by WU-Blast-2 to maximize the alignmentscore are ignored).

The term “positives”, in the context of sequence comparison performed asdescribed above, includes residues in the sequences compared that arenot identical but have similar properties (e.g. as a result ofconservative substitutions). The % value of positives is determined bythe fraction of residues scoring a positive value in the BLOSUM 62matrix divided by the total number of residues in the longer sequence,as defined above.

In a similar manner, “percent (%) nucleic acid sequence identity” isdefined as the percentage of nucleotides in a candidate sequence thatare identical with the nucleotides in the UCP5 coding sequence. Theidentity values can be generated by the BLASTN module of WU-BLAST-2 setto the default parameters, with overlap span and overlap fraction set to1 and 0.125, respectively.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the UCP5 naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” nucleic acid molecule encoding a UCP5 polypeptide is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the UCP5-encoding nucleic acid. An isolatedUCP5-encoding nucleic acid molecule is other than in the form or settingin which it is found in nature. Isolated nucleic acid moleculestherefore are distinguished from the UCP5-encoding nucleic acid moleculeas it exists in natural cells. However, an isolated nucleic acidmolecule encoding a UCP5 polypeptide includes UCP5-encoding nucleic acidmolecules contained in cells that ordinarily express UCP5 where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers single anti-UCP5 monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies) and anti-UCP5 antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C. followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10° dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a UCP5 polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of UCP5which retain the biologic and/or immunologic activities of native ornaturally-occurring UCP5. A preferred activity is the ability to affectmitochondrial membrane potential in a way that results in an up- ordown-regulation of metabolic rate and/or heat production. One suchactivity includes the generation of proton leakage in mitochondrialmembrane that results in an increase in metabolic rate.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native UCP5 polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native UCP5polypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, or fragments or amino acid sequence variants of native UCP5polypeptides.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cows, horses, sheep, pigs, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

II. Compositions and Methods of the Invention

A. Full-Length UCP5

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas UCP5. In particular, cDNA encoding a UCP5 polypeptide has beenidentified and isolated, as disclosed in further detail in the Examplesbelow. For sake of simplicity, in the present specification the proteinencoded by DNA 80562-1663 as well as all further native homologues andvariants included in the foregoing definition of UCP5, will be referredto as “UCP5,”, regardless of their origin or mode of preparation.

As disclosed in the Examples below, a clone DNA 80562-1663 has beendeposited with ATCC and assigned accession no. 203325. The actualnucleotide sequence of the clone can readily be determined by theskilled artisan by sequencing of the deposited clone using routinemethods in the art. The predicted amino acid sequence can be determinedfrom the nucleotide sequence using routine skill. For the UCP5 herein,Applicants have identified what is believed to be the reading frame bestidentifiable with the sequence information available at the time offiling.

Using Align software (GNE), it has been found that a full-length nativesequence UCP5 (shown in FIG. 1 and SEQ ID NO: 1) has about 38% aminoacid sequence identity with UCP3, about 36% amino acid sequence identitywith UCP2, and about 33% amino acid sequence identity with UCP1.Accordingly, it is presently believed that UCP5 disclosed in the presentapplication is a newly identified member of the human uncoupling proteinfamily and may possess activity(s) and/or property(s) typical of thatprotein family, such as the ability to enhance or supress metabolic rateby affecting mitochondrial membrane potential.

B. UCP5 Variants

In addition to the full-length native sequence UCP5 polypeptidesdescribed herein, it is contemplated that UCP5 variants can be prepared.UCP5 variants can be prepared by introducing appropriate nucleotidechanges into the UCP5 DNA, and/or by synthesis of the desired UCP5polypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the UCP5, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence UCP5 or in various domainsof the UCP5 described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the UCP5 that results in a change in the amino acidsequence of the UCP5 as compared with the native sequence UCP5.Optionally the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of the UCP5.Guidance in determining which amino acid residue may be inserted,substituted or deleted without adversely affecting the desired activitymay be found by comparing the sequence of the UCP5 with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of 1 to 5 amino acids. The variation allowed may be determined bysystematically making insertions, deletions or substitutions of aminoacids in the sequence and, if desired, testing the resulting variantsfor activity in assays known in the art or as described herein.

One embodiment of the invention is directed to UCP5 variants which arefragments of the full length UCP5. Preferably, such fragments retain adesired activity or property of the full length UCP5.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the UCP5 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of UCP5

Covalent modifications of UCP5 are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a UCP5 polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of the UCP5. Derivatization with bifunctional agentsis useful, for instance, for crosslinking UCP5 to a water-insolublesupport matrix or surface for use in the method for purifying anti-UCP5antibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxy-succinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the UCP5 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence UCP5 (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequenceUCP5. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the UCP5 polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence UCP5 (for O-linkedglycosylation sites). The UCP5 amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the UCP5 polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theUCP5 polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the UCP5 polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzmmol., 138:350 (1987).

Another type of covalent modification of UCP5 comprises linking the UCP5polypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The UCP5 of the present invention may also be modified in a way to forma chimeric molecule comprising UCP5 fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theUCP5 with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the UCP5. The presence ofsuch epitope-tagged forms of the UCP5 can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe UCP5 to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the UCP5 with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a UCP5 polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

The UCP5 of the invention may also be modified in a way to form achimeric molecule comprising UCP5 fused to a leucine zipper. Variousleucine zipper polypeptides have been described in the art. See, e.g.,Landschulz et al., Science, 240:1759 (1988); WO 94/10308; Hoppe et al.,FEBS Letters, 344:1991 (1994); Maniatis et al., Nature, 341:24 (1989).Those skilled in the art will appreciate that the leucine zipper may befused at either the 5′ or 3′ end of the UCP5 molecule.

D. Preparation of UCP5

The description below relates primarily to production of UCP5 byculturing cells transformed or transfected with a vector containing UCP5nucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare UCP5. Forinstance, the UCP5 sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of theUCP5 may be chemically synthesized separately and combined usingchemical or enzymatic methods to produce the full-length UCP5.

1. Isolation of DNA Encoding UCP5

DNA encoding UCP5 may be obtained from a cDNA library prepared fromtissue believed to possess the UCP5 mRNA and to express it at adetectable level. Accordingly, human UCP5 DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The UCP5-encoding gene may also be obtainedfrom a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the UCP5 oroligonucleotides of at least about 20-80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding UCP5 is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra, and are described above in SectionI.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as BLAST, BLAST2, ALIGN, DNAstar, and INHERIT to measure identityor positives for the sequence comparison.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein, and, if necessary, using conventional primer extensionprocedures as described in Sambrook et al., supra, to detect precursorsand processing intermediates of mRNA that may not have beenreverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for UCP5 production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and KS 772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forUCP5-encoding vectors. Saccharomyces cerevisiae is a commonly used lowereukaryotic host microorganism.

Suitable host cells for the expression of glycosylated UCP5 are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells. Examples of useful mammalian host cell lines include Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding UCP5 may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The UCP5 may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe UCP5-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov.

1990. In mammalian cell expression, mammalian signal sequences may beused to direct secretion of the protein, such as signal sequences fromsecreted polypeptides of the same or related species, as well as viralsecretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2 μm plasmid origin is suitable for yeast,and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) areuseful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theUCP5-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the UCP5-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding UCP5.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Req., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

UCP5 transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the UCP5 by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theUCP5 coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding UCP5.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of UCP5 in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceUCP5 polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to UCP5DNA and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of UCP5 may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of UCP5 can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify UCP5 from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of theUCP5. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular UCP5 produced.

E. Uses for UCP5

Nucleotide sequences (or their complement) encoding UCP5 have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. UCP5 nucleic acid will also beuseful for the preparation of UCP5 polypeptides by the recombinanttechniques described herein.

The full-length native sequence UCP5 gene (SEQ ID NO: 2), or fragmentsthereof, may be used as, among other things, hybridization probes for acDNA library to isolate the full-length UCP5 gene or to isolate stillother genes (for instance, those encoding naturally-occurring variantsof UCP5 or UCP5 from other species) which have a desired sequenceidentity to the UCP5 sequence disclosed in FIG. 1 (SEQ ID NO: 1).Optionally, the length of the probes will be about 20 to about 80 bases.The hybridization probes may be derived from the nucleotide sequence ofSEQ ID NO: 2 or from genomic sequences including promoters, enhancerelements and introns of native sequence UCP5. By way of example, ascreening method will comprise isolating the coding region of the UCP5gene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the UCP5 gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Fragments of UCP5 DNA contemplated by the invention include sequencescomprising at least about 20 to 30 consecutive nucleotides of the DNA ofSEQ ID NO: 2. Preferably, such sequences comprise at least about 50consecutive nucleotides of the DNA of SEQ ID NO: 2.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related UCP5 coding sequences.

Nucleotide sequences encoding a UCP5 can also be used to constructhybridization probes for mapping the gene which encodes that UCP5 andfor the genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for UCP5 encode a protein which binds toanother protein, the UCP5 can be used in assays to identify the otherproteins or molecules involved in the binding interaction. By suchmethods, inhibitors of the receptor/ligand binding interaction can beidentified. Proteins involved in such binding interactions can also beused to screen for peptide or small molecule inhibitors or agonists ofthe binding interaction. Also, the receptor UCP5 can be used to isolatecorrelative ligand(s). Screening assays can be designed to find leadcompounds that mimic the biological activity of a native UCP5 or areceptor for UCP5. Such screening assays will include assays amenable tohigh-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

Nucleic acids which encode UCP5 or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding UCP5 can be used to clone genomic DNA encodingUCP5 in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding UCP5. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for UCP5 transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding UCP5 introduced into the germline of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding UCP5. Such animals can beused as tester animals for reagents thought to confer protection from,for example, pathological conditions associated with its overexpressionor underexpression. In accordance with this facet of the invention, ananimal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of UCP5 can be used to construct aUCP5 “knock out” animal which has a defective or altered gene encodingUCP5 as a result of homologous recombination between the endogenous geneencoding UCP5 and altered genomic DNA encoding UCP5 introduced into anembryonic cell of the animal. For example, cDNA encoding UCP5 can beused to clone genomic DNA encoding UCP5 in accordance with establishedtechniques. A portion of the genomic DNA encoding UCP5 can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors). The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras. [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the UCP5polypeptide.

Nucleic acid encoding the UCP5 polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

It is believed that the UCP5 gene therapy has applications in, forinstance, treating metabolic conditions. This can be accomplished, forexample, using the techniques described above and by introducing a viralvector containing a UCP5 gene into certain tissues (like muscle or fat)to increase metabolic rate in these targeted tissues and thereby elevateenergy expenditure.

Generally, methods of treatment employing UCP5 are contemplated by theinvention. Fuel combustion, electron transport, proton pumping and O₂consumption (which may be referred to collectively as metabolic rate)are coupled to ATP synthesis. There can be an “inefficiency” in mammals,such that a portion of metabolic rate (in some cases which may begreater than 20%) may be ascribed to H⁺ “leak” back into the matrixspace with no ATP synthesis.

It is believed UCP5 may be involved in catalyzing H⁺ leak, therebyplaying a role in energetic inefficiency in vivo. Accordingly,modulating UCP5 activity or quantities (presence or expression) of UCP5in mammalian tissues (particularly, metabolically important tissues),may concomitantly modulate H⁺ leak, metabolic rate and heat production.The methods of modulating (either in an up-regulation or down-regulationmode) metabolic rate in a mammal has a variety of therapeuticapplications, including treatment of obesity and the symptoms associatedwith stroke, trauma (such as burn trauma), sepsis and infection.

In the treatment of obesity, those skilled in the art will appreciatethat the modulation of mitochonrial membrane potential may be used toincrease body metabolic rate, thereby enhancing an individual's abilityfor weight loss. Screening assays may be conducted to identify moleculeswhich can up-regulate expression or activity (such as the uncoupling) ofUCP5. The molecules thus identified can then be employed to increasemetabolic rate and enhance weight loss.

UCP5 may also be employed in diagnostic methods. For example, thepresence or absence of UCP5 activity, or alternatively over- orunder-expression of UCP5 in an individual's cells, can be detected. Theskilled practitioner may use information resulting from such detectionassays to assist in predicting metabolic conditions or risk for onset ofobesity. If it is determined, for instance, that UCP5 activity in apatient is abnormally high or low, therapy such as hormone therapy orgene therapy could be administered to return the UCP5 activity orexpression to a physiologically acceptable state.

Detection of impaired UCP5 function in the mammal may also be used toassist in diagnosing impaired neural activity or neural degeneration. Itis presently believed UCP5 may be involved in the regulation of braintemperature or metabolic rate that is required for normal brain function(and associated neural activity). It is also presently believed thatUCP5 may control the generation of reactive oxygen species and thereforecontribute to neural degeneration. Molecules identified in the screeningassays which have been found to suppress UCP5 expression or function mayalso be employed to treat fever since it is believed that UCP5 isup-regulated during episodes of fever.

UCP5 has been found to be expressed in a relatively wide number oftissues and is believed to be involved in the maintenance of metabolicrate in mammals. As described in the Examples section of theapplication, isoforms of UCP5 are differentially expressed in humantissues and have different levels of activities in modulatingmitochondrial membrane potential. An alteration of UCP5 expression orrelative abundance of its isoforms in mammalian tissue(s) may lead to analteration in metabolic rate (for instance, a lower or decreasedexpression of UCP5 or an alteration of UCP5 tissue distribution may bepresent in obese mammals). Such alteration in expression or distributionof UCP5 isoforms may also result in a predisposition to obesity inmammals.

Accordingly, the UCP5 molecules described in the application will beuseful in diagnostic methods. For example, the presence or absence ofUCP5 activity, or alternatively over- or under-expression, in anindividual's cells or tissues, can be detected using assays known in theart, including those described in the Examples below. The inventionprovides a method of detecting expression of UCP5 (or its isoforms) in amammalian cell or tissue sample, comprising contacting a mammalian cellor tissue sample with a DNA probe and analyzing expression of UCP5 mRNAtranscript in said sample. Quantitative RT-PCR methods using DNA primersand probes which are isoform specific may also be employed to assist inquantitating specific isoform mRNA abundance. Further, DNA arraytechnologies in the art may be employed to quantitate one or moreisoform(s) RNA abundance. The sample may comprise various mammaliancells or tissues, including but not limited to, liver tissue, whiteadipose tissue and skeletal muscle. The skilled practitioner may useinformation resulting from such detection assays to assist in predictingmetabolic conditions or onset of obesity. If it is determined, forinstance, that UCP5 expression (or abundance) levels or distributionlevels in a patient are abnormally high or low as compared to a controlpopulation of mammals of corresponding age and normal body weight (oralternatively, to a population of mammals diagnosed as being obese),therapy such as gene therapy, diet control, etc. may be employed totreat the mammal.

Detection of impaired UCP5 expression or function in the mammal may alsobe used to assist in diagnosing or treating impaired neural activity orneural degeneration. It is known in the art that reactive oxygen speciescan cause cellular damage in various tissues, particularly in braintissue, and more particularly in brain neuronal tissue. An increase inthe presence or generation of reactive oxygen species has beenassociated with Down's syndrome, as well as other neurodegenerativediseases. It is believed that UCP5 or its isoforms can regulate thegeneration of reactive oxygen species and may play a protective role.

Accordingly, in the treatment of the conditions described above, thoseskilled in the art will appreciate that the modulation of UCP5expression or activity may be used to, for instance, increase bodymetabolic rate, thereby enhancing an individual's ability for weightloss. Screening assays may be conducted to identify molecules which canup-regulate expression or activity (such as the uncoupling) of UCP5. Themolecules thus identified can then be employed to increase metabolicrate and enhance weight loss. The UCP5 polypeptides are useful in assaysfor identifying lead compounds for therapeutically active agents thatmodulate expression or activity of UCP5. Candidate molecules orcompounds may be assayed with the mammals' cells or tissues to determinethe effect(s) of the candidate molecule or compound on UCP5 expressionor activity. Such screening assays may be amenable to high-throughputscreening of chemical libraries, and are particularly suitable foridentifying small molecule drug candidates. Small molecules include butare not limited to synthetic organic or inorganic compounds. The assayscan be performed in a variety of formats, including protein-proteinbinding assays, biochemical screening assays, immunoassays, cell basedassays, etc. Such assay formats are well known in the art.

Accordingly, in one embodiment, there is provided a method of conductinga screening assay to identify a molecule which enhances or up-regulateseither activity and/or expression of UCP5, comprising the steps ofexposing a mammalian cell or tissue sample believed to comprise UCP5 toa candidate molecule and subsequently analyzing expression and/oractivity of UCP5 in said sample. In this method, the sample may befurther analyzed for mitochondrial membrane potential. Optionally, theUCP5 is a native polypeptide or any of the specific isoforms of UCP5identified herein. The sample being analyzed may comprise variousmammalian cells or tissues, including but not limited to human braintissue. The screening assay may be an in vitro or in vivo assay. By wayof example, an in vivo screening assay may be conducted in a transgenicanimal wherein a promoter for a UCP5 gene may be linked to a reportergene such as luciferase or beta-galactosidase. Optionally, “knock in”technology may be used in this regard in which such a reporter gene isinserted 5 to the promoter (which in turn is linked to a genomicsequence encoding a UCP5). Such techniques are known in the art. Thecandidate molecule employed in the screening assay may be a smallmolecule comprising a synthetic organic or inorganic compound. In analternative embodiment, the screening assay is conducted to identify amolecule which decreases or down-regulates activity and/or expression ofUCP5. The effect(s) that such candidate molecule may have on theexpression and/or activity of UCP5 may be compared to a control orreference sample, such as for instance, expression or activity of UCP5observed in a like mammal.

F. Anti-UCP5 Antibodies

The present invention further provides anti-UCP5 antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-UCP5 antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the UCP5 polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-UCP5 antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the UCP5 polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against UCP5.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-UCP5 antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe UCP5, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymoloqy, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

G. Uses for Anti-UCP5 Antibodies

The anti-UCP5 antibodies of the invention have various utilities. Forexample, anti-UCP5 antibodies may be used in diagnostic assays for UCP5,e.g., detecting its expression in specific cells or tissues. Variousdiagnostic assay techniques known in the art may be used, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-UCP5 antibodies also are useful for the affinity purification ofUCP5 from recombinant cell culture or natural sources. In this process,the antibodies against UCP5 are immobilized on a suitable support, sucha Sephadex resin or filter paper, using methods well known in the art.The immobilized antibody then is contacted with a sample containing theUCP5 to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the UCP5, which is bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the UCP5 from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

EXAMPLE 1 Isolation of cDNA clones Encoding Human UCP5

EST databases, including public EST databases (e.g., GenBank), weresearched for sequences having homologies to human UCP3. The search wasperformed using the computer program BLAST or BLAST2 [Altschul et al.,Methods in Enzymology, 266:460-480 (1996)] as a comparison of the UCP3protein sequences to a 6 frame translation of the EST sequences. Thosecomparisons resulting in a BLAST score of 70 (or in some cases, 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program AssemblyLIGN and MacVector(Oxford Molecular Group, Inc.).

A DNA sequence (“from DNA”) was assembled relative to other ESTsequences using AssemblyLIGN software (FIG. 5; SEQ ID NO: 5). ESTs fromthe GenBank and Merck databases used in the assembly included thesequences having the following accession nos.: R19440; AA15735; R44688;AA142931; N48177; AA056945; AA021118; AA054608; AA401224; N53324;AA057005; AA015832; AA404241; AI032869; AA910774; AI131262; AI128486;AI241428; AA021119; and AI039086. In addition, the from DNA sequence wasextended using repeated cycles of BLAST and AssemblyLIGN to extend thesequence as far as possible using the sources of EST sequences discussedabove.

Based on this DNA sequence, oligonucleotides were synthesized to isolatea clone of the full-length coding sequences for UCP5 by PCR. Forward andreverse PCR primers generally range from 20 to 30 nucleotides and areoften designed to give a PCR product of about 100-1000 bp in length. Theprobe sequences are typically 40-55 bp in length. In some cases,additional oligonucleotides are synthesized when the consensus sequenceis greater than about 1-1.5 kbp.

PCR primers (forward and reverse) were synthesized: (SEQ ID NO: 3)forward PCR primer GAACTGGCAAGATCCTGCTACCC (A-381V) (SEQ ID NO: 4)reverse PCR primer GCTGGCAGGGCTGGGCTCAC (A-381W)

RNA for construction of the cDNA libraries was isolated from human Bcell, fetal kidney, and substantia nigra tissues, as well as mousehypothalamus. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

Corresponding full length cDNAs were obtained by polymerase chainreaction (PCR) from the human substantia nigra library and the mousehypothalamus cDNA library, and cloned into a mammalian expression vectorpRK7 (Genentech, Inc). Eight to ten clones from each library weresequenced, among which it was noted that different clones encodedmultiple isoforms.

DNA sequencing of the clones isolated by PCR as described above gave thefull-length DNA sequence for human UCP5 (designated herein as DNA80562-1663 [FIG. 1, SEQ ID NO: 2]) and the derived protein sequence forUCP5 (FIG. 1, SEQ ID NO: 1). DNA sequences of what are believed to betwo other isoforms of the human UCP5 gene, hUCP5S [FIG. 12, SEQ ID NO:6] and hUCP5SI [FIG. 13, SEQ ID NO: 8], and two isoforms, mUCP5S [FIG.14, SEQ ID NO: 10] and mUCP5L [FIG. 15, SEQ ID NO: 12], of the mouseUCP5 gene were similarly identified. An alignment of the derived aminoacid sequences for UCP5 (SEQ ID NO: 1), hUCP5S (SEQ ID NO: 7), hUCP5SI(SEQ ID NO: 9), mUCP5S (SEQ ID NO: 11) and mUCP5L (SEQ ID NO: 13) isshown in FIG. 16.

The entire coding sequence of the full length human UCP5 is shown inFIG. 1 (SEQ ID NO: 2). Clone DNA 80562-1663 contains a single openreading frame with an apparent translational initiation site atnucleotide positions 10-12, and an apparent stop codon at nucleotidepositions 985-987. (See FIG. 1; SEQ ID NO: 2). The predicted polypeptideprecursor is 325 amino acids long. It is presently believed that UCP5 isa membrane-bound protein and contains at least 6 transmembrane regions.MacVector software (Oxford Molecular Group, Inc.) was used to identifyprotein features. Transmembrane domain regions were identified at theamino acid sequence of residues 42 to 61, 103 to 117, 142 to 160, 202 to218, 236 to 255, and 294 to 317 (using the numbering of amino acidresidues according to FIG. 1 (SEQ ID NO:1)). These putativetransmembrane regions in the UCP5 amino acid sequence are illustrated inFIG. 2.

The following additional features also were identified. A signal peptidewas identified at the amino acid sequence of residues 1 to 19. Atyrosine kinase phosphorylation site was identified at the amino acidsequence of residues 78 to 84. Thirteen N-myristoylation sites werefound at the amino acid sequence starting at residues 2, 47, 86, 106,123, 148, 152, 178, 195, 199, 246, 249, and 278. Three mitochondrialcarrier protein motifs were identified at the amino acid sequence ofresidues 60 to 68, 161 to 169, and 255 to 263. A unique hydrophobicamino terminal sequence (amino acids 1-23) which may be involved inmembrane anchoring was also identified.

hUCP5S is shorter than UCP5, when aligned with UCP5 (FIG. 16), hUCP5Sappears to be identical except that three amino acids (amino acids23-25, as shown in FIG. 16) in its unique amino terminal portion aredeleted. hUCP5SI as compared to UCP5 has a 31-amino acid insertionbetween transmembrane domains III and IV and lacks the three amino acidresidues, 23-25 of FIG. 1 (see FIG. 16). This insertional sequence inhUCP5SI also contains a hydrophobic segment that may also be involved ininteraction with the mitochondrial membrane. The hUCP5 and hUCP5Sprotein sequences appear to be highly conserved with the mouse sequence,with only 8 conserved amino acid changes (FIG. 16).

Clone DNA 80562, designated as DNA 80562-1663, contained in the pcDNA3vector (Invitrogen) has been deposited with ATCC and is assigned ATCCdeposit No. 203325. UCP5 polypeptide is obtained or obtainable byexpressing the molecule encoded by the cDNA insert of the deposited ATCC203325 vector. Digestion of the vector with BamHI and EcoRI restrictionenzymes will yield an approximate 972 plus 34 bp insert. The full-lengthUCP5 protein shown in FIG. 1 has an estimated molecular weight of about36,202 daltons and a pI of about 9.88.

An alignment of the amino acid sequence of UCP5 with UCPs 1, 2, 3, and 4is illustrated in FIG. 2. The human UCP5 gene has been mapped tochromosome X (q23-q25).

EXAMPLE 2 A. Northern Blot Analysis

Expression of UCP5 mRNA in human and mouse tissues was examined byNorthern blot analysis. Human and mouse RNA blots were hybridized to a 1kilobase ³²P-labelled DNA probe based on the full length UCP5 cDNA; theprobe was generated by digesting pcDNA3UCP5 (for the human blots) orpRRTmouseUCP5 (for the mouse blots) and purifying the UCP5 cDNA insert.Human adult RNA blot MTN-II (Clontech) (FIGS. 3A, 3B, 3C), PBLs (FIG.3B), and cancer cells (FIG. 3D) were incubated with the DNA probes. Asshown in FIG. 3D, the cancer cells probed included HL-60 (promyelocyticleukemia), HeLa cells, K562 (chronic myelogenous leukemia), MOLT-4(lymphoblastic leukemia), Raji (Burkitt's lymphoma), SW480 (colorectaladenocarinoma), A549 (lung carcinoma), and G361 (melanoma). Two humanbrain multiple tissue Northern blots (Clontech) and a mouse multipletissue Northern blot (Clontech) were also similarly probed with humanUCP5 and mouse UCP5 cDNA probes, respectively. The blots weresubsequently probed with a β-actin cDNA.

Northern analysis was performed according to manufacturer's instructions(Clontech). The blots were developed after overnight exposure to x-rayfilm.

As shown in FIG. 3, UCP5 mRNA transcripts were detected. Two UCP5 mRNAtranscripts (approximately 1.7 and 2.4 kb) were detected in multiplehuman tissues and cancer cells_(FIGS. 3A-D). Relatively high levels oftranscript were present in human testis, brain, and heart. FurtherNorthern blot analysis using two multiple tissue blots revealed thatUCP5 transcript (1.7 kb) was present in most regions of the brain, withlow levels found in spinal cord and corpus callosum (FIGS. 3E and 3F).When a mouse multiple tissue Northern blot was analyzed, mUCP5transcripts were similarly detected in heart, brain, liver, kidney andtestis (FIG. 3G).

B. Real Time Quantative RT-PCR

Total tissue RNA was extracted from various mouse tissues discussedbelow using total RNA Isolation reagent (Biotecx Lab, Inc., Houston,Tex.) according to the manufacturer's instructions. For real timeRT-PCR, the extracted RNA was then treated with Dnase I (GIBCO BRL) toremove DNA contained in the extract. Gene expression analysis for UCP5was performed as described in King, K. L. et al., Endocrine, 9:45-55(1998) and Gibson, U. E. M. et al., Genome Res., 6:995-1001 (1996).Primers and probes were designed using Primer Express Software (PEApplied Biosciences, Foster City, Calif.).

For mUCP5L: (SEQ ID NO: 14) forward primer, 5′-AAA TTT GCA ACG GCGGC-3′; (SEQ ID NO: 15) reverse primer, 5′-TCA GAC CAG ACA TTT CAT GGC T-3′; (SEQ ID NO: 16) probe, 5′ (FAM)-TGA TTG TAA GCG GAC ATC AGA AAA GTTCCA CTT T-(TAMARA)3′.

For total mouse UCP5: (SEQ ID NO: 17) Forward primer, 5′-GGG TGT GGT CCCAAC TGC T-3′; (SEQ ID NO: 18) Reverse primer, 5′TTC TTG GTA ATA TCA TAAACG GGC A-3′; (SEQ ID NO: 19) probe, 5′ (FAM)- CGT GCT GCA ATC GTT GTGGGA GTA GAG-(TAMARA)3′.

For mouse beta-actin: (SEQ ID NO: 20) forward primer, 5′-GAA ATC GTG CGTGAC ATC AAA GAG-3′; (SEQ ID NO: 21) reverse primer, 5′-CTC CTT CTG CATCCT GTC AGC AA-3′; (SEQ ID NO: 22) probe, 5′(FAM)-CGG TTC CGA TGC CCTGAG GCT C (TAMARA)-3′.

For UCP5: (SEQ ID NO: 23) forward primer, 5′-GGA ATA ATC CTA AAT TTT CTAAGG GTG A-3′; (SEQ ID NO: 24) reverse primer, 5′-CTT TTC TGG TGT CCG CTTACA A- 3′; (SEQ ID NO: 25) probe, 5′ (FAM)-TTT GCA ACG GCG GCCGTG-(TAMARA) 3′.

For hUCP5SI: (SEQ ID NO: 26) forward primer, 5′-GGC TCT GTG GAG GTG CTTATG-3′; (SEQ ID NO: 27) reverse primer, 5′-TGG GAT TAC AGG CAT GAGCC-3′; (SEQ ID NO: 28) probe, 5′ (FAM)-CAA AAG CTG TTA CCG GCT GTG TGCTG-(TAMARA)3′

For total human UCP5: (SEQ ID NO: 29) forward primer, 5′-GGA TGT TCC ATGCGC TGT T-3′; (SEQ ID NO; 30) reverse primer, 5′-CGC AGG AGC AAT TCC TGAA-3′; (SEQ ID NO: 31) probe, 5′ (FAM)-CGC ATC TGT AAA GAG GAA GGT GTATTG GCT CTC-(TAMARA)3′.

The thermal cycling conditions were as follows: 15 min at 50° C. and 10min at 95° C., followed by 40 cycles of 95° C. for 15 sec and 60° C. for1 min. All reactions were performed using Model 7700 Sequence Detector(PE Applied Biosciences). β-actin was used to normalize for differencesin the amount of mRNA in each reaction, as its abundance was notaffected by treatments. Each RNA sample was run in duplicate and themean values of the duplicates were used to calculate the gene expressionlevel.

For determination of tissue distribution of UCP5 in human tissues, totalRNA from various human tissues (Clontech) was analyzed by a real timequantitative RT-PCR assay, with 18S rRNA used as a normalization control(primers and probes purchased from PE Applied Biosciences). The relativeabundance of hUCP5S was obtained by subtraction of the UCP5 level fromthe total UCP5 level.

Consistent with the Northern blot analyses, abundant UCP5 mRNA wasdetected in human brain, testis, kidney, uterus, heart, lung, stomach,liver, and skeletal muscle, with the greatest expression in brain andtestis (FIG. 3H). In mouse, UCP5 was detected in brain, testis, liver,white adipose tissue, brown adipose tissue, kidney, skeletal muscle andheart, with mUCP5S being the predominant form (FIG. 3I). The relativeabundance of UCP5 and UCP5S in brain is dramatically different betweenhuman and mouse. Generally, UCP5 is more abundant in human than in mousetissue, ranging from 12% (kidney) to 100% (brain) of the total UCP5mRNA. Human skeletal muscle had approximately equal amounts of UCP5L andUCP5S. UCP5L is the predominant form in human brain, while 98% of theUCP5 mRNA is UCP5S in mouse brain. Further, UCP5S was predominant in allthe other tissues examined. For example, 85% of the UCP5 mRNA is UCP5Sin human liver, and UCP5L is detectable only in mouse brain and whiteadipose tissue (FIGS. 3H and 3I) A trace amount of UCP5SI was present inhuman substantia nigra and hippocampus, but was undetectable in allother tissues.

EXAMPLE 3 Use of UCP5 as a Hybridization Probe

The following method describes use of a nucleotide sequence encodingUCP5 as a hybridization probe.

DNA comprising the coding sequence of full-length or mature UCP5 (asshown in FIG. 1, SEQ ID NO: 2) is employed as a probe to screen forhomologous DNAs (such as those encoding naturally-occurring variants ofUCP5) in human tissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled UCP5-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence UCP5 can then be identified using standardtechniques known in the art.

EXAMPLE 4 Expression of UCP5 in E. coli

This example illustrates preparation of UCP5 by recombinant expressionin E. coli.

The DNA sequence encoding UCP5 (SEQ ID NO: 2) is initially amplifiedusing selected PCR primers. The primers should contain restrictionenzyme sites which correspond to the restriction enzyme sites on theselected expression vector. A variety of expression vectors may beemployed. An example of a suitable vector is pBR322 (derived from E.coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will optionally includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the UCP5 coding region,lambda transcriptional terminator, and an argu gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. If no signal sequence is present, and theexpressed UCP5 is intracellular, the cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized UCP5 protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein. If a signal sequence is present, the expressed UCP5 can beobtained from the cell's periplasm or culture medium. Extraction and/orsolubilization of the UCP5 polypeptides can be performed using agentsand techniques known in the art. (See e.g. U.S. Pat. Nos. 5,663,304;5,407,810).

EXAMPLE 5 Expression of UCP5 in Mammalian Cells

This example illustrates preparation of UCP5 by recombinant expressionin mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the UCP5 DNA is ligated into pRK5with selected restriction enzymes to allow insertion of the UCP5 DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-UCP5.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-UCP5 DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of UCP5 polypeptide. The cultures containing transfected cellsmay undergo further incubation (in serum free medium) and the medium istested in selected bioassays.

In an alternative technique, UCP5 may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-UCP5 DNA is added.The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed UCP5 can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, UCP5 can be expressed in CHO cells. The pRK5-UCP5can be transfected into CHO cells using known reagents such as CaPO₄ orDEAE-dextran. As described above, the cell cultures can be incubated,and the medium replaced with culture medium (alone) or medium containinga radiolabel such as ³⁵S-methionine. After determining the presence ofUCP5 polypeptide, the culture medium may be replaced with serum freemedium. Preferably, the cultures are incubated for about 6 days, andthen the conditioned medium is harvested. The medium containing theexpressed UCP5 can then be concentrated and purified by any selectedmethod.

Epitope-tagged UCP5 may also be expressed in host CHO cells. The UCP5may be subcloned out of the pRK5 vector. The subclone insert can undergoPCR to fuse in frame with a selected epitope tag such as a poly-his taginto a Baculovirus expression vector. The poly-his tagged UCP5 insertcan then be subcloned into a SV40 driven vector containing a selectionmarker such as DHFR for selection of stable clones. Finally, the CHOcells can be transfected (as described above) with the Sv40 drivenvector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedUCP5 can then be concentrated and purified by any selected method, suchas by Ni²⁺-chelate affinity chromatography.

In an alternative method, the UCP5 may be expressed intracellularly(where no signal sequence is employed). This intracellular expression,and subsequent extraction or solubilization and purification may beperformed using techniques and reagents known in the art.

EXAMPLE 6 Expression of UCP5 in Yeast

The following method describes recombinant expression of UCP5 in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of UCP5 from the ADH2/GAPDH promoter. DNAencoding UCP5 and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof UCP5. For secretion, DNA encoding UCP5 can be cloned into theselected plasmid, together with DNA encoding the ADH2/GAPDH promoter, anative UCP5 signal peptide or other mammalian signal peptide, or, forexample, a yeast alpha-factor or invertase secretory signal/leadersequence, and linker sequences (if needed) for expression of UCP5.Alternatively, the native signal sequence of UCP5 is employed.

Yeast cells, such as S. cerevisiae yeast strain AB110, can then betransformed with the expression plasmids described above and cultured inselected fermentation media as set forth, for instance, in U.S. Pat.Nos. 4,775,662 and 5,010,003. The transformed yeast supernatants can beanalyzed by precipitation with 10% trichloroacetic acid and separationby SDS-PAGE, followed by staining of the gels with Cobmassie Blue stain.

Recombinant UCP5 can subsequently be isolated and purified by removingthe yeast cells from the fermentation medium by centrifugation and thenconcentrating the medium using selected cartridge filters. Theconcentrate containing UCP5 may further be purified using selectedcolumn chromatography resins. In an alternative method, the UCP5 may beexpressed intracellularly (where no signal sequence is employed). Theintracellular expression, and subsequent extraction or solubilizationand purification may be performed using techniques and reagents known inthe art.

EXAMPLE 7 Expression of UCP5 in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of UCP5 inBaculovirus-infected insect cells.

The sequence coding for UCP5 is fused upstream of an epitope tagcontained within an expression vector. Such epitope tags includepoly-his tags and immunoglobulin tags (like Fc regions of IgG). Avariety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding UCP5 or the desired portion of the coding sequence ofUCP5 is amplified by PCR with primers complementary to the 5′ and 3′regions, The 5′ primer may incorporate flanking (selected) restrictionenzyme sites. The product is then digested with those selectedrestriction enzymes and subcloned into the expression vector. The vectormay contain the native signal sequence for UCP5 if secretion is desired.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged UCP5 can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged UCP5 are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) UCP5 can beperformed using known chromatography techniques, including for instance,Protein A or protein G column chromatography.

EXAMPLE 8 Measurement of Mitochondrial Membrane Potential Change Inducedby UCP5

Assays were conducted to determine the effects of UCP5 expression onmitochondrial membrane potential.

Human embryonic kidney 293 cells (ATCC CCL 1573) were grown in culturemedium (DMEM, 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mlpenicillin, 100 microgram/ml streptomycin) to 60§-80% confluence in100-mm plates and co-transfected with 1-1.5 μg pGreen Lantern-1(GibcoBRL) and 7.5 μg UCP5, UCP3-expressing constructs or vector controlplasmid using Fugene™ 6 transfection reagent (Boehringer Mannheim;according to manufacturer's instructions). The transfected cells wereharvested 24 hours post-transfection and resuspended in 1 ml culturemedium containing 150 ng/ml TMRE (tetramethylrhodamine ethyl ester) andincubated for 30 minutes at 37° C. in the dark. The cells were thenwashed with 2 ml culture medium, resuspended in 1 ml culture medium andanalyzed by flow cytometry. The transfected cells were identified basedon the expression of fluorescence protein (GFP). Analyses of the sampleswere performed on an EPICS Elite-ESP (Beckman-Coulter). Samples wereanalyzed utilizing two spatially separated lasers. The primary laser wasan argon-ion laser with fluorescence excitation at 531 nm. Fluorescenceemission was detected at 525 nm and 575 nm, respectively. Approximately10,000 cells were analyzed for each sample.

The results are illustrated in FIGS. 4A-4F. Expression of UCP3 in the293 cells reduced mitochondrial membrane potential (mmp) by 45% (n=6;[+SD]=2.3%) (FIGS. 4B and 4E) in comparison to the controlvector-transfected cells (FIGS. 4C and 4D). Expression of UCP5 in the293 cells reduced mmp by 30% (n=6; [±SD]=2%) (FIGS. 4A and 4F).

UCP3 was localized to the mitochondrial membrane and an NH₂-Flag tag didnot affect its uncoupling activity or mitochondrial localization [Mao,W. et al, FEBS Lett. 443:326-330 (1999)]. In contrast, an NH₂-tagcompletely abolished the uncoupling activity of UCP5L and itsmitochondrial localization.

FIG. 17 shows the ability of different isoforms of UCP5 to reducemembrane potential. Expression of hUCP5S in the 293 cells significantlyreduced mmp, but not to the extent of hUCP5L (FIG. 17C). A similarobservation was made for mUCP5L and mUCP5S (FIG. 17C). hUCP5SI showed anactivity comparable to that of hUCP5L (FIG. 17C). The expression of UCP5isoforms in these transfected cells were monitored by a real timequantitative RT-PCR assay, as described above, and no differences wereobserved.

EXAMPLE 9 Preparation of Antibodies that Bind UCP5

This example illustrates preparation of monoclonal antibodies which canspecifically bind UCP5.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified UCP5, fusion proteins containing UCP5, andcells expressing recombinant UCP5 on the cell surface. Selection of theimmunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the UCP5 immunogen emulsifiedin complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-UCP5 antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of UCP5. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstUCP5. Determination of “positive” hybridoma cells secreting the desiredmonoclonal antibodies against UCP5 is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-UCP5monoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

EXAMPLE 10 The Expression of UCP5 mRNA in Mice Subjected to Food andTemperature Stresses

To evaluate whether UCP5 has uncoupling activity in situ important tometabolism, the amount of UCP5 mRNA produced in tissues of mice thatwere subjected to food and temperature stresses, i.e., metabolicchallenges, was determined. Depending on the role UCP5 may have inmetabolism, the amount of UCP5 mRNA produced in a tissue may vary withstresses to metabolism such as fasting, fat consumption, and exposure totemperature below room temperature.

The mice in this study were fed normal rodent chow (Purina Rodent Chow5010; Purina, St. Louis, Mo.) and water ad libitum unless indicatedotherwise. The type of mouse studied varied depending on the conditionused to challenge the metabolism of the mouse studied and will bedescribed below.

Generally, the mice studied were exposed to light 12 hours a day from6:00 a.m. until 6:00 p.m. at which time they were exposed to dark forthe following 12 hours.

The mice were sacrificed under CO₂ just prior to tissue harvest, whichoccurred in the morning between 8:00 and 12:00 a.m. unless specifiedotherwise. The tissues were harvested and total tissue RNA was preparedusing reagents and protocols from Biotecx Lab, Houston, Tex. Although anumber of tissues were collected from each mouse, the study focused onmeasuring the abundance of UCP5 mRNA in the brain (because the brain hashigh UCP5 gene expression) and in the liver (because the liver isimportant to energy expenditure and metabolism). At least 5mice/treatment were used in the studies.

Real time quantitative reverse-transcriptase polymerase chain reaction(RT-PCR), as described above, was used to determine the amount of UCP5mRNA in the harvested tissues. RT-PCR was performed using mRNA samples.[Heid et al., Genome Research, 6:986-994 (1996); Gibson et al., GenomeResearch, 6:995-1001 (1996)]. Generally, to carry out real timequantitative RT-PCR, primers and probes specific to UCP5 were used(TaqMan Instrument, PE Biosciences, Foster City, Calif.). Valves werecorrected for mRNA loading using β-actin mRNA abundance as loadingcontrol. The following primers and probes were used:

For liver UCP5: (SEQ ID NO: 17) forward primer: 5′GGG TGT GGT CCC AACTGC T3′; (SEQ ID NO: 18) reverse primer: 5′TTC TTG GTA ATA TCA TAA ACGGGC A3′; (SEQ ID NO: 19) probe: 5′ (FAM) CGT GCT GCA ATC GTT GTG GGA GTAGAG(TAMARA)3′.

For beta-actin: (SEQ ID NO: 20) forward primer: 5′GAA ATC GTG CGT GACATC AAA GAG 3′; (SEQ ID NO: 21) reverse primer: 5′CTC CTT CTG CAT CCTGTC AGC AA 3′; (SEQ ID NO: 22) probe: 5′ (FAM) CGG TTC CGA TGC CCT GAGGCT C (TAMARA)3′.The Effect of Food Consumption on UCP5 mRNA Expression

In a first study, seven-week old male mice (CS7BL/6J; Bar Harbor, Me.)were studied to evaluate the effect of fasting and eating meals on UCP5mRNA production in the mice studied. The mice were obtained at six weeksof age and at seven weeks were randomly assigned to one of three groups:control mice fed ad lib, mice fasted for 24 hours, and mice fasted for24 hours and then fed ad lib for 24 hours.

The mice were sacrificed as described above after ad lib feeding for thefirst group, after 24 hours of fasting for the second group, and afterthe 48 hours of first fasting and then ad lib feeding for the thirdgroup. The tissues were harvested as described above.

Quantitative RT-PCR was performed for brain and liver tissues accordingto the methods described above and the amount of UCP5 mRNA produced inthe brain and liver was quantified. Statistical differences across thegroups were determined using a protected Fisher's least significantdifference analysis (L. Ott, An Introduction to Statistical Methods andData Analysis, 3rd Ed., Boston: PWS-Kent Publishing Co., 1988). The datapresented in FIGS. 6A to 6C and 7A to 7C represent means +/−SEM. Anasterisk indicates a statistical difference of at least p<0.05.

The results obtained for the brain tissue are illustrated in FIGS. 6A to6C, and the results obtained for the liver tissue are illustrated inFIGS. 7A to 7C.

FIGS. 6A and 7A illustrate the UCP5 mRNA abundance in the brain tissueand liver tissue, respectively, from mice that were fed ad lib. FIGS. 6Band 7B illustrate the UCP5 mRNA abundance in the brain tissue and livertissue, respectively, from mice that fasted for 24 hours. FIGS. 6C and7C illustrate the UCP5 mRNA abundance in the brain tissue and livertissue, respectively, from mice that fasted for 24 hours and then werefed ad lib for 24 hours.

Typically, fasting and restriction of food consumption lower metabolicrate, suggesting that expression of UCP5 mRNA would decrease for micethat were fasting compared to mice that were fed ad lib. FIG. 7Bindicates a decrease in UCP5 mRNA expression in liver tissue for themice that fasted compared to the mice that were fed ad lib as shown inFIG. 7A or the mice that were fed after fasting as shown in FIG. 7C.

The Effect of Fat Consumption on UCP5 mRNA Expression

In a second study, four-week old male mice (A/J or C57BL/6J; JacksonLabs, Bar Harbor, Me.) were studied to evaluate the effect of high andlow fat diets on UCP5 mRNA production in the mice studied.

The mice were obtained at four weeks of age and immediately placed oneither a low fat diet or high fat diet (Research Diets, Inc., NewBrunswick, N.J.) patterned after those formulated by Surwit et al.,Metabolism, 44(5): 645-651 (1995), containing 11% or 58% fat (%calories), respectively. Animals were fed ad lib for approximately threeweeks (days 22-23 on diet). They were then sacrificed, and their tissueswere harvested as described above. Quantitative RT-PCR was performed forthe brain and liver tissue according to the methods described above andthe amount of UCP5 mRNA produced in the brain and liver tissues wasquantified. Statistical differences across the groups were determinedusing a protected Fisher's least significant difference analysis (L.Ott, An Introduction to Statistical Methods and Data Analysis, 3rd Ed.,Boston: PWS-Kent Publishing Co., 1988). The data presented in FIGS. 8Ato 8D and 9A to 9D represent means +/−SEM. An asterisk indicates astatistical difference of at least p<0.05.

The results obtained for the brain tissue are illustrated in FIGS. 8A to8D, and the results obtained for the liver tissue are illustrated inFIGS. 9A to 9D.

FIGS. 8A and 9A illustrate the UCP5 mRNA abundance in brain and livertissue, respectively, from A/J mice that were fed a low fat diet, andFIGS. 8B and 9B illustrate the UCP5 mRNA abundance in brain tissue andliver tissue, respectively, from A/J mice that were fed a high fat diet.FIGS. 8C and 9C illustrate the UCP5 mRNA abundance in brain tissue andliver tissue, respectively, from C57BL6/J mice that were fed a low fatdiet. FIGS. 8D and 9D illustrate the UCP5 mRNA abundance in brain tissueand liver tissue, respectively, from C57BL6/J mice that were fed a highfat diet.

A/J mice have been shown to be “obesity-resistant” on a high fat dietcompared to “obesity-prone” C57BL6/J (see Surwit et al., supra). Thismay be due to a lower metabolic efficiency in the A/J strain—i.e., theyapparently put on fewer calories per calories ingested. FIG. 9Bindicates an increase in UCP5 mRNA expression in liver tissue from A/Jmice fed a high fat diet compared to A/J mice fed a low fat diet asshown in FIG. 9A. Similar results for liver UCP5 mRNA expression werenot obtained for the “obesity prone” C57BL6/J mice (FIGS. 9C and 9D),and similar results were not obtained for the brain tissue from eitherA/J mice (FIGS. 8A and 8B) or C57BL6/J mice (FIGS. 8C and 8D).

The Effect of Temperature Stress on UCP5

In a third study, male mice (FVB-N; Taconic, Germantown, N.Y.) werestudied to evaluate the effect of exposing the mice to temperaturestresses. Typically, cold exposure in rodents elicits an increase inmetabolic rate. This metabolic increase may be to support a stable bodytemperature. Yet warm-acclimation, which is defined as chronic exposureto temperatures within the murine thermoneutral zone (approx. 30-35°C.), lowers metabolic rate. [Klaus et al., Am. J. Physiol.,274:R287-R293 (1998)].

The mice in this study were housed two per cage and were randomlyassigned to the following groups: a control group (housed at 22° C. for3 weeks), a warm-acclimated group (housed at 33° C. for 3 weeks), afood-restricted group (housed at 22° C. for 3 weeks, but given accesseach day to the average amount of food eaten by warm-acclimated mice theday before), a cold-challenged group (housed at 22° C. for 3 weeks priorto the initiation of exposure to 4° C.). For the cold-challenged mice,beginning in the morning, mice were exposed to 4° C. by being placedinto a 4° C. room for 1, 6, 24, or 48 hours prior to sacrificing themice and harvesting the tissue.

The mice were sacrificed and tissues were harvested at six week of ageas described above. Quantitative RT-PCR was performed for the brain andliver tissues according to the methods described above and the amount ofUCP5 mRNA produced in the brain and liver tissues was quantified.Statistical differences across the groups were determined using aprotected Fisher's least significant difference analysis (L. Ott, AnIntroduction to Statistical Methods and Data Analysis, 3rd Ed., Boston:PWS-Kent Publishing Co., 1988). The data presented in FIGS. 10A to 10Gand 11A to 11G represent +/−SEM. An asterisk indicates a statisticaldifference of at least p<0.05.

The results obtained for the brain tissue are illustrated in FIGS. 10Ato 10G, and the results obtained for the liver tissue are illustrated inFIGS. 11A to 11G.

FIGS. 10A and 11A illustrate the UCP5 mRNA abundance in the controlgroup of mice. FIGS. 10B to 10E and 11B to 11E illustrate the UCP5 mRNAabundance in the brain tissue and liver tissue, respectively, from thegroup of mice that were cold-challenged for 1, 6, 24, and 48 hours,respectively. FIGS. 10F and 11F illustrate the UCP5 mRNA abundance inthe brain tissue and liver tissue, respectively, from thefood-restricted group of mice, and FIGS. 10G and 11G illustrate the UCP5mRNA abundance in the brain tissue and liver tissue, respectively, fromthe warm-acclimated group of mice.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC): Material ATCC Dep. No. Deposit Date DNA80562-1663 203325Oct. 6, 1998

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC '122 and the Commissioner's rules pursuantthereto (including 37 CFR '1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated UCP5 polypeptide comprising a polypeptide having at leastan 80% sequence identity to the sequence of amino acid residues fromabout 20 to about 325 of FIG. 1 (SEQ ID NO: 1).
 2. The isolatedpolypeptide of claim 1 comprising amino acid residues from about 1 toabout 325 of FIG. 1 (SEQ ID NO: 1).
 3. An isolated UCP5 polypeptidescoring at least 80% positives when compared to the sequence of aminoacid residues from about 1 to about 325 of FIG. 1 (SEQ ID NO: 1).
 4. Anisolated UCP5 polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, and SEQ ID NO: 13, or a fragment thereof sufficient toprovide a binding site for an anti-UCP5 antibody.
 5. An isolated UCP5polypeptide encoded by the cDNA insert of the vector deposited as ATCCDeposit No. 203325 (DNA 80562-1663).
 6. An isolated polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a UCP5 polypeptide comprising the sequenceof amino acid residues from about 1 to about 325 of FIG. 1 (SEQ ID NO:1), or (b) the complement of the DNA molecule of (a), and, if said testDNA molecule has at least about an 80% sequence identity to (a) or (b),(ii) culturing a host cell comprising said test DNA molecule underconditions suitable for the expression of said polypeptide, and (iii)recovering said polypeptide from the cell culture.
 7. A chimericmolecule comprising a UCP5 polypeptide fused to a heterologous aminoacid sequence.
 8. The chimeric molecule of claim 7, wherein saidheterologous amino acid sequence is an epitope tag sequence.
 9. Thechimeric molecule of claim 7, wherein said heterologous amino acidsequence is a Fc region of an immunoglobulin.